Detection of microsatellite instability and its use in diagnosis of tumors

ABSTRACT

Methods and kits are disclosed for use in the analysis of microsatellite instability in genomic DNA. Methods and kits are also disclosed which can be used to detect microsatellite instability DNA present in biological materials, such as tumors. The methods and kits of the present invention can be used to detect or diagnose diseases associated with microsatellite instability, such as certain types of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/314,810, filed Dec. 9, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/841,366, filed on Apr. 24, 2001, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.09/663,020, filed Sep. 15, 2000, now U.S. Pat. No. 6,844,152.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made using U.S. government Small Business InnovationResearch Program Grant CA76834-02 from the National Institutes ofHealth. The U.S. government retains certain rights to the invention.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the detection of instability in regions ofgenomic DNA containing simple tandem repeats, such as microsatelliteloci. The invention particularly relates to multiplex analysis for thepresence or absence of instability in a set of microsatellite loci ingenomic DNA from cells, tissue, or bodily fluids originating from atumor. The invention also relates to the use of microsatelliteinstability analysis in the detection and diagnosis of cancer andpredisposition for cancer.

BACKGROUND OF THE INVENTION

Microsatellite loci of genomic DNA have been analyzed for a wide varietyof applications, including, but not limited to, paternity testing,forensics work, and in the detection and diagnosis of cancer. Cancer canbe detected or diagnosed based upon the presence of instability atparticular microsatellite loci that are unstable in one or more types oftumor cells.

A microsatellite locus is a region of genomic DNA with simple tandemrepeats that are repetitive units of one to five base pairs in length.Hundreds of thousands of such microsatellite loci are dispersedthroughout the human genome. Microsatellite loci are classified based onthe length of the smallest repetitive unit. For example, loci withrepetitive units of 1 to 5 base pairs in length are termed“mononucleotide”, “dinucleotide”, “trinucleotide”, “tetranucleotide”,and “pentanucleotide” repeat loci, respectively.

Each microsatellite locus of normal genomic DNA for most diploidspecies, such as genomic DNA from mammalian species, consists of twoalleles at each locus. The two alleles can be the same or different fromone another in length and can vary from one individual to the next.Microsatellite alleles are normally maintained at constant length in agiven individual and its descendants; but, instability in the length ofmicrosatellites has been observed in some tumor types (Aaltonen et al.,1993, Science 260:812-815; Thibodeau et al., 1993 Science 260:816-819;Peltomaki et al., 1993 Cancer Research 53:5853-5855; Ionov et al., 1993Nature 363:558-561). This form of genomic instability in tumors, termedmicrosatellite instability (hereinafter, “MSI”), is a molecular hallmarkof the inherited cancer syndrome Hereditary Nonopolyposis ColorectalCancer (hereinafter, “HNPCC”). The cause of MSI in HNPCC is thought tobe a dysfunctional DNA mismatch repair system that fails to reverseerrors that occur during DNA replication (Fishel et al., 1993 Cell75:1027-38; Leach et al., 1993 Cell 75:215-25; Bronner et al., 1994Nature 368:258-61; Nicolaides et al., 1994 Nature 371:75-80; Miyaki etal., 1997 Nat Genetics 17:271-2). Insertion or deletion of one or morerepetitive units during DNA replication persists without mismatch repairand can be detected as length polymorphisms by comparison of allelesizes found in microsatellite loci amplified from normal and tumor DNAsamples (Thibodeau et al., 1993, supra).

MSI has been found in over 90% of HNPCC and in 10-20% of sporadiccolorectal tumors (Liu et al., 1996 Nature Med 2:169-174; Thibodeau etal., 1993, supra; Ionov et al., 1993 Nature 363:558-561; Aaltonen etal., 1993 Science 260: 812-816; Lothe et al., 1993 Cancer Res. 53:5849-5852). However, MSI is not limited to colorectal tumors. MSI hasalso been detected in pancreatic cancer (Han et al., 1993 Cancer Res53:5087-5089) gastric cancer (Id.; Peltomaki et al., 1993 Cancer Res53:5853-5855; Mironov et al., 1994 Cancer Res 54:41-44; Rhyu et al.,1994 Oncogene 9:29-32; Chong et al., 1994 Cancer Res 54:4595-4597),prostate cancer (Gao et al., 1994 Oncogene 9:2999-3003), endometrialcancer (Risinger et al., 1993 Cancer Res 53:5100-5103; Peltomaki et al.,1993 Cancer Res 53:5853-5855), and breast cancer (Patel et al., 1994Oncogene 9:3695-3700).

The genetic basis of HNPCC is thought to be a germ-line mutation in oneof several DNA mismatch repair genes (hereinafter “MMR”) (Leach et al.,1993 Cell 75:1215-1225; Fishel et al., 1993 Cell 75:1027-38; Leach etal., 1993 Cell 75:215-25; Bronner et al., 1994 Nature 368:258-61;Nicolaides et al., 1994 Nature 371:75-80; Miyaki et al., 1997 NatGenetics 17:271-2; Papadopoulos et al., 1994 Science 263:1625-1629)Among HNPCC patients, 50-60% have been reported to carry inheritedmutations in two mismatch repair genes, MSH2 and MLH1 (Kolodner et al.,1999 Cancer Research 59:5068:5074). Moreover, 70-100% of HNPCC caseswhose tumors manifest a high frequency MSI (hereinafter “MSI-H”)phenotype reportedly have germ-line mutations in these two genes. Fewgerm-line mutations in MSH6, MSH3, PMS1 and PMS2 genes have beenreported in HNPCC patients, indicating that inherited mutations in thesemismatch repair genes play a minor role in HNPCC (Peltomaki et al., 1997Gastroenterologly 113:1146-1158; Liu et al., 1996 Nat Med 2:169-174;Kolodner et al., 1999 Cancer Research 59:5068-5074). Without functionalrepair proteins, errors that occur during replication are not repairedleading to high mutation rates and increased likelihood of tumordevelopment.

Repetitive DNA is particularly sensitive to errors in replication andtherefore dysfunctional mismatch repair systems result in widespreadalterations in microsatellite regions. A study of yeast cells withoutfunctional mismatch repair systems showed a 2800, 284, 52, and 19 foldincrease in mutation rates in mono-, di-, tri-, tetra-, andpentanucleotide repeats, respectively (Sia et al., 1997 Molecular andCellular Biology 17:2851-2858). Mutations in mismatch repair genes arenot thought to play a direct role in tumorigenesis, but rather act byallowing DNA replication errors to persist. Mismatch repair deficientcells have high mutation rates and if these mutations occur in genesinvolved in tumorigenesis the result can lead to the development ofcancer. MSI positive tumors have been found to carry somatic frameshiftmutations in mononucleotide repeats in the coding region of severalgenes involved in growth control, apoptosis, and DNA repair (e.g.,TGFBRII, BAX, IGFIIR, TCF4, MSH3, MSH6) (Planck et al., 2000 Genes,Chromosomes & Cancer 29:33-39; Yamamoto et al., 1998 Cancer Research58:997-1003; Grady et al., 1999 Cancer Research 59:320-324; Markowitz etal., 1995 Science 268:1336-1338; Parsons et al., 1995 Cancer Research55:5548-5550). The most commonly altered locus is TGFBRII, in which over90% of MSI-H colon tumors have been found to contain a mutation in the10 base polyadenine repeat present in the gene (Markowitz et al., 1995Science 268:1336-1338).

MSI occurs in almost all HNPCC tumors regardless of which mismatchrepair gene is involved. MSI has also been shown to occur early intumorigenesis. These two factors contribute to making MSI analysis anexcellent diagnostic test for the detection of HNPCC. In addition, MSIanalysis can serve as a useful pre-screening test to identify potentialHNPCC patients for further genetic testing. MSI analysis of sporadiccolorectal carcinomas is also desirable, since the occurrence of MSIcorrelates with a better prognosis (Bertario et al., 1999 InternationalJ Cancer 80:83-7).

One long-standing problem with diagnosing HNPCC is that colon tumorbiopsies from a person with HNPCC look the same pathologically as asporadic colon tumor, making diagnosis of the syndrome difficult. Sinceprognosis, therapy and follow-up will be different for patients withHNPCC, it is important to find more definitive diagnostic methods.However, mutation detection in HNPCC patients remains difficult becausethere are at least 5 known MMR genes which are large genes without knownhot spots for mutations. Direct gene sequencing remains the most precisemethod of mutation detection, but is time consuming and expensive(Terdiman et al., 1999 The American Journal of Gastroenterology94:23544-23560). In addition, high sensitivity and specificity can bedifficult to obtain with sequencing alone because many mutations thatare detected may be harmless polymorphisms that have no affect on thefunction of the mismatch repair proteins.

DNA analysis of microsatellite loci makes it theoretically possible todevelop a blood test for use in the detection of specific types ofcancer. Early studies have shown that tumor DNA is released into thecirculation, and is present in particularly high concentrations inplasma and serum in a number of different types of cancer (Leon et al.,1977 Cancer Res 37:646-650; Stroun et al., 1989 Oncology 46:318-322).Since then, DNA released into the blood from several different types oftumors has been detected by analysis of microsatellite DNA using thepolymerase chain reaction (hereinafter, “PCR”) (Hibi et al., 1998 CancerResearch 58:1405-1407; Chen et al., 1999 Clinical Cancer Research5:2297-2303; Kopreski et al., 1999 Clinical Cancer Research 5:1961-1965;Fujiwara et al., 1999 Cancer Research 59:1567-1571; Chen et al., 1996Nature Medicine 2:1033-1034; Goessl et al., 1998 Cancer Research58:4728-4732; Miozzo et al., 1996 Cancer Research 56:2285-2288).

The first tumor-specific gene sequences detected in blood from patientswith cancer were mutated K-ras genes (Vasioukhin et al., 1994 Br. J.Haematol 86: 774-779; Sorenson et al., 1994 Cancer Epidemiol. Biomark.Prev. 3:67-71; Sorenson et al., 2000 Clinical Cancer Research6:2129-2137; Anker et al., 1997 Gastroenterology 112:1114-1120). Morerecently, detection of microsatellite instability in soluble tumor DNAfrom plasma and serum originating from head and neck squamous cellcancers (Nawroz et al., 1996 Nature Med 2:1035-1037) and small cell lungcancers (Chen et al., 1996 Nature Med 2:1033-1035) has been shown. Thesesuccesses have stimulated searches for microsatellite instability incirculating tumor DNA from many other cancer types. Hibi et al., usedmicrosatellite markers to search for the presence of genetic alterationsin serum DNA from colon cancer patients (Hibi, K. et al., 1998 CancerResearch 58:1405-1407). Hibi et al., also reported that eighty percentof primary tumors in the colon cancer patients displayed MSI and/or lossof heterozygosity (hereinafter, “LOH”), another type of mutationdiscussed below. No microsatellite or LOH mutations were detected inpaired serum DNA. However, identical K-ras mutations were found incorresponding tumor and serum DNAs, indicating that tumor DNA waspresent in the blood. (Id.)

The detection of circulating tumor cells and micrometastases may alsohave important prognostic and therapeutic implications. Becausedisseminated tumor cells are present in very small numbers, they are noteasily detected by conventional immunocytological tests, which can onlydetect a single tumor cell among 10,000 to 100,000 normal cells(Ghoussein et al., 1999 Clinical Cancer Research 5:1950-1960). Moresensitive molecular techniques based on PCR amplification oftumor-specific abnormalities in DNA or RNA have greatly facilitateddetection of occult (hidden) tumor cells. PCR-based tests capable ofroutinely detecting one tumor cell in one million normal cells have beendevised for identification of circulating tumor cells andmicrometastases in leukemias, lymphomas, melanoma, neuroblastoma, andvarious types of carcinomas. (Id.)

Most targets for detection of disseminated tumor cells have been mRNAs.However, some DNA targets have been used successfully, including K-rasmutations in colon cancers, as noted above. The presence ofmicrosatellite instability in some types of tumor cells raises thepossibility that these tumor specific mutations created by theinstability could serve as a target for PCR-based detection of occulttumor cells.

There has been considerable controversy about how to precisely defineand accurately measure MSI (Boland, 1998 Cancer Research 58:5248-5257).Reports on the frequency of MSI in various tumors ranges considerably.For example, different studies have reported ranges of 3% to 95% MSI forthe frequency of MSI in bladder cancer (Gonzalez-Zulueta et al., 1993Cancer Research 53:28-30; Mao et al., 1996 PNAS 91:9871-9875). Oneproblem with defining MSI is that it is both tumor specific and locusdependent (Boland et al. 1998, supra). Thus, the frequency of MSIobserved with a particular tumor type in a single study will depend onthe number of tumors analyzed, the number of loci investigated, how manyloci need to be altered to score a tumor as having MSI and whichparticular loci were included in the analysis. To help resolve theseproblems, the National Cancer Institute sponsored a workshop on MSI toreview and unify the field (Id.). As a result of the workshop a panel offive microsatellites was recommended as a reference panel for futureresearch in the field. This panel included two mononucleotide lociBAT-25, BAT-26, and three dinucleotide loci D5S346, D2S123, D17S250.

One particular problem in MSI analysis of tumor samples occurs when oneof the normal alleles for a given marker is missing due to LOH, and noother novel fragments are present for that marker (Id.). One cannoteasily discern whether this represents true LOH or MSI in which theshifted allele has co-migrated with the remaining wild-type allele. Incases like this, the recommendation from the NCI workshop on MSI was notto call it as MSI. One way to minimize this type of problem would be touse loci that displayed low frequency of LOH in colon tumors.

Clinical diagnostic assays used for determining treatment and prognosisof disease require that the tests be highly accurate (low falsenegatives) and specific (low false positive rate). Many informativemicrosatellite loci have been identified and recommended for MSI testing(Boland et al. 1998, supra). However, even the most informativemicrosatellite loci are not 100% sensitive and 100% specific. Tocompensate for the lack of sensitivity using individual markers,multiple markers can be used to increase the power of detection. Theincreased effort required to analyze multiple markers can be offset bymultiplexing. Multiplexing allows simultaneous amplification andanalysis of a set of loci in a single tube and can often reduce thetotal amount of DNA required for complete analysis. To increase thespecificity of an MSI assay for any given type of cancer, it has beenrecommended that the panel of five highly informative microsatelliteloci identified at the National Institute Workshop (see above) bemodified to substitute or add other loci of equal utility (Boland et al.1998, supra, at p. 5250). Increased information yielded from amplifyingand analyzing greater numbers of loci results in increased confidenceand accuracy in interpreting test results.

Multiplex MSI analysis solves problems of accuracy and discrimination ofMSI phenotypes, but the additional complexity can make analysis morechallenging. For example, when microsatellite loci are co-amplified andanalyzed in a multiplex format, factors affecting ease and accuracy ofdata interpretation become much more essential. One of the primaryfactors affecting accurate data interpretation is the amount of stutterthat occurs at microsatellite loci during PCR (Bacher & Schumm, 1998Profiles in DNA 2:3-6; Perucho, 1999 Cancer Research 59:249-256).Stutter products are minor fragments produced by the PCR process thatdiffer in size from the major allele by multiples of the core repeatunit. The amount of stutter observed in microsatellite loci tends to beinversely correlated with the length of the core repeat unit. Thus,stutter is most severely displayed with mono- and dinucleotide repeatloci, and to a lesser degree with tri-, tetra-, and pentanucleotiderepeats (Bacher & Schumm, 1998, supra). Use of low stutter loci inmultiplexes would greatly reduce this problem. However, carefulselection of loci is still necessary in choosing low stutter locibecause percent stutter can vary considerably even within a particularrepeat type (Micka et al., 1999 Journal of Forensic Sciences 44:1-15).

Microsatellite multiplex systems have been primarily developed for usein genotyping, mapping studies and DNA typing applications. Thesemultiplex systems are designed to allow co-amplification of multiplemicrosatellite loci in a single reaction, followed by detection of thesize of the resulting amplified alleles. For DNA typing analysis, theuse of multiple microsatellite loci dramatically increases the matchingprobability over a single locus. Matching probability is a commonstatistic used in DNA typing that defines the number of individuals youwould have to survey before you would find the same DNA pattern as arandomly selected individual. For example, a four locus multiplex system(GenePrint™ CTTv Multiplex System, Promega) has a matching probabilityof 1 in 252.4 in African-American populations, compared to an eightlocus multiplex system (GenePrint™ PowerPlex™ 1.2 System, Promega) whichhas a matching probability of 1 in 2.74×10⁸ (Proceedings: AmericanAcademy of Forensic Sciences (Feb. 9-14, 1998), Schumm, James W. et al.,p. 53, B88; Id. Gibson, Sandra D. et al., p. 53, B89; Id., Lazaruk,Katherine et al., p. 51, B83; Sparkes, R. et al., 1996 Int J Legal Med109:186-194). Other commercially available multiplex systems for DNAtyping include AmpF/STR Profiler™ and AmpF/STR COfiler™ (AmpFISTRProfiler™ PCR Amplification Kit User's Manual (1997), i-viii and 1-1 to1-10; and AmpFISTR COfiler™ PCR Amplification Kit User Bulletin (1998),i-iii and 1-1 to 1-10, both published by Perkin-Elmer Corp). In additionto multiplexes for DNA typing, a few multiplex microsatellite systemshave been developed for the detection of diseases, such as cancer. Onesuch system has been developed by Roche Diagnostics, the “HNPCCMicrosatellite Instability Test”, in which five MSI loci (BAT25, BAT26,D5S436, D17S250, and D2S123) are co-amplified and analyzed. Additionalsystems are needed, particularly systems that include additional locidisplaying high sensitivity to MSI and low stutter for easy and accuracyof analysis.

The materials and methods of the present invention are designed for usein multiplex analysis of particular microsatellite loci of human genomicDNA from various sources, including various types of tissue, cells, andbodily fluids. The present invention represents a significantimprovement over existing technology, bringing increased power ofdiscrimination, precision, and throughput to the analysis of MSI lociand to the diagnosis of illness, such as cancer, related to mutations atsuch loci.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and kits for amplifying andanalyzing microsatellite loci or sets of microsatellite loci. Thepresent invention also provides methods and kits for detecting cancer inan individual by co-amplifying multiple microsatellite loci of humangenomic DNA originating from tumor tissue or cancerous cells.

In one aspect, the present invention provides a method of analyzingmicro-satellite loci, comprising: (a) providing primers forco-amplifying in a single tube a set of at least three microsatelliteloci of genomic DNA, comprising at least one mononucleotide repeat locusand at least two tetranucleotide repeat loci; (b) co-amplifying the setof at least three microsatellite loci from a sample of genomic DNA in amultiplex amplification reaction, using the primers, thereby producingamplified DNA fragments; and (c) determining the size of the amplifiedDNA fragments.

In another aspect, the present invention provides a method ofco-amplifying the set of at least three microsatellite loci of at leasttwo different samples of genomic DNA, a first sample originating fromnormal non-cancerous biological material from an individual and a secondsample originating from a second biological material from theindividual. The at least two samples of human genomic DNA areco-amplified in separate multiplex amplification reactions, usingprimers to each of the loci in the set of at least three microsatelliteloci. The size of the resulting amplified DNA fragments from the twomultiplex reactions are compared to one another to detect instability inany of the at least three microsatellite loci of the second sample ofhuman genomic DNA.

Another embodiment of the present invention is a method of analyzing atleast one mononucleotide repeat locus of human genomic DNA selected fromthe group consisting of MONO-11 and MONO-15. The method of analyzing theat least one mononucleotide repeat locus selected from the groupconsisting of MONO-11 and MONO-15 comprises the steps of: (a) providingat least one primer of the at least one mononucleotide repeat locus; (b)amplifying the at least one mononucleotide repeat locus from a sample ofgenomic DNA originating from a biological material from an individualhuman subject, using the at least one primer, thereby producing anamplified DNA fragment; and (c) determining the size of the amplifiedDNA fragments. The amplified DNA fragments are preferably analyzed todetect microsatellite instability at the at least one mononucleotiderepeat locus by comparing the size of the amplified DNA fragments to themost commonly observed allele size at that locus in a human population.Alternatively, the method is used to amplify the at least onemononucleotide repeat locus of a sample of human genomic DNA from normalnon-cancerous biological material from an individual, and microsatelliteinstability is detected by comparing the resulting amplified DNAfragments to those obtained in step (b).

Another embodiment of the present invention is a kit for the detectionof microsatellite instability in DNA isolated from an individualsubject, comprising a single container with oligonucleotide primers forco-amplifying a set of at least three microsatellite loci comprising onemononucleotide locus and two tetranucleotide loci.

The various embodiments of the method and kit of the present invention,described briefly above, are particularly suited for use in thedetection of MSI in tumor cells or cancerous cells. Specifically, themethod or kit of the present invention can be used to amplify at leastone mononucleotide repeat locus selected from the group consisting ofMONO-11 and MONO-15 or the set of at least three microsatellite locicomprising at least one mononucleotide repeat locus and at least twotetranucleotide repeat loci of at least one sample of genomic DNA frombiological material, such as tissue or bodily fluids, preferablybiological material containing or suspected of containing DNA fromtumors or cancerous cells. For monomorphic or quasi-monomorphic loci,such as MONO-11 and MONO-15, one can compare the resulting pattern tothe pattern produced by amplifying normal DNA from any individual in apopulation with a standard pattern at that locus. However, it ispreferable to use DNA from normal tissue of the same individual fromwhom the tumor DNA was obtained, in order to ensure that a positiveresult does not reflect a germline mutation, rather than MSI.

The method and kit can also be used to compare the results of multiplexamplification of DNA from normal tissue of an individual to the resultsof multiplex amplification of DNA from other biological material fromthe same individual. Use of this particular embodiment of the method ofthe present invention to detect MSI in tumor cells by comparison tonormal cells is illustrated in FIG. 1. Specifically, FIG. 1 shows atetranucleotide repeat (GATA), amplified by a primer pair (“primer A”and “primer B”) in a polymerase chain reaction (“PCR”), followed byseparation of amplified alleles by size using capillary electrophoresis,and a plot of the fractionated amplified alleles using GeneScan™software. Note that only the two alleles and small stutter peaks appearin the plot of amplified DNA from normal DNA, while three MSI peaksappear in addition to the two allele peaks in the plot of amplifiedtumor DNA.

Advantages and a fuller appreciation of the specific attributes of thisinvention will be gained upon an examination of the following figures,detailed description of preferred embodiments, and appended claims. Itis expressly understood that the drawings are for the purposes ofillustration and description only, and are not intended as a definitionof the limits of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Illustration of microsatellite instability analysis. The figureis a diagram of a primer pair annealed to a tetranucleotide locus on twoalleles of the same genomic DNA, and plots of results of capillaryelectrophoresis of products of amplification of a tetranucleotide locusof DNA originating from normal vs. tumor tissue. MSI peaks are indicatedin the plot of amplified DNA from tumor tissue.

FIG. 2. Illustration of effect of microsatellite repeat unit length onamount of stutter observed. The figure includes a diagram of a primerpair annealed to a tetranucleotide repeat locus on two different allelesof genomic DNA, and a set of fluorescent scans and plots of amplifiedmono-, di-, tri-, tetra-, and pentanucleotide repeat loci from humangenomic DNA from various individuals, amplified and fractionated by gelor by capillary electrophoresis.

FIG. 3. Demonstration that low stutter tetranucleotide repeat loci areeasier to interpret than high stutter dinucleotide repeat loci. Thefigure is a plot of results of capillary electrophoresis of products ofthe amplification of two tetranucleotide and two dinucleotide repeatloci of two different sets of samples of DNA originating from normal vs.tumor tissue.

FIG. 4. Illustration of variance in amount of stutter within selectedtetranucleotide and pentanucleotide repeat loci. The figure is a plot ofthe variability in percent stutter observed in a 13 differenttetranucleotide and 5 different pentanucleotide repeat loci. The boxesrepresent the average percent stutter and the solid bars the range ofstutter observed for each locus.

FIG. 5. Results of screening of tetranucleotide repeat markers forfrequency of microsatellite instability. The figure is a plot of thenumber of microsatellite loci, out of a total of 273 markers, thatdisplays a given percent MSI. For example, approximately 15 loci werealtered in 100% of MSI-H tumor samples evaluated.

FIG. 6. Results of screening of pentanucleotide repeat markers forfrequency of microsatellite instability. The figure is a plot of thepercent MSI observed for each of eight different tetranucleotide repeatloci in a set of nine MSI-H and a set of 30 MSS tumors.

FIG. 7. Microsatellite instability analysis using MONO-15 marker. Thefigure is a plot generated from capillary electrophoresis products ofamplification of the MONO-15 locus of DNA from four different sets ofpaired normal and tumor samples originating from four differentindividuals.

FIG. 8. Percent MSI in 59 colon cancer samples using nine-locus MSImultiplex. The figure is a plot of the percent MSI observed in 59 coloncancer samples (29 MSH and 30 MSI-L or MSS samples) using the nine locusMSI multiplex described in Example 6, below (i.e., D1S518, D3S2432,D7S1808, D7S3046, D7S9070, D10S1426, BAT-25, BAT-26, and MONO-15).

FIG. 9. Fluorescent multiplex microsatellite analysis using a nine-locusMSI Multiplex. The figure is a plot generated from capillaryelectrophoresis of products of multiplex amplification of normalnon-cancerous human genomic, using the nine locus MSI multiplex used inFIG. 8.

FIG. 10. Detection of microsatellite instability in colon cancer samplesusing a nine-locus MSI multiplex. The figure is a plot generated fromcapillary electrophoresis of products of multiplex amplification of DNAfrom paired normal and colon tumor sample, using the nine locus MSImultiplex used in FIG. 8.

FIG. 11. Detection of microsatellite instability in colon cancer samplesusing nine-locus MSI multiplex is the same type of plot shown in FIG.10, generated using a different sample of paired normal and colon cancerDNA from a different individual.

FIG. 12. Detection of microsatellite instability in stomach cancersamples using nine-locus MSI multiplex. The figure is a plot generatedfrom capillary electrophoresis of products of multiplex amplification ofDNA from paired normal and stomach cancer tumor samples, using the ninelocus MSI multiplex described in FIG. 8.

FIG. 13. Microsatellite analysis of paraffin embedded tissues withnine-locus MSI multiplex. The figure is a plot generated from capillaryelectrophoresis of products of multiplex amplification of DNA fromparaffin embedded tissue, using the nine locus MSI multiplex describedin FIG. 8.

FIG. 14. Percent MSI in colon cancer samples at 10 differentmicrosatellite loci. The figure is a bar graph of the percent MSIobserved in 66 colon cancer samples (36 MSI-H and 30 MSI stable or MSI-Lsamples) using a nine loci contained in the MSI multiplex described inExample 8 (i.e., BAT-26, D7S3070, D7S3046, BAT-40, MONO-15, D7S1808,BAT-25, D10S1426 and D3S2432) and D1S518.

FIG. 15. Fluorescent multiplex microsatellite analysis using anine-locus MSI Multiplex. The figure is a plot generated from capillaryelectrophoresis of products of multiplex amplification of normalnon-cancerous human genomic DNA, using the nine-locus MSI multiplexdescribed in Example 8 labeled with primers labeled with fluorescentdyes, as follows. Primers to BAT-26, D7S3070, and D7S3046 were labeledwith fluorescein; primers to BAT-40, MONO-15 and D7S1808 were labeledwith JOE; and primers to BAT-25, D10S1426 and D3S2432 were labeled withTMR.

FIG. 16. Detection of microsatellite instability in colon cancer samplesusing a nine-locus MSI multiplex. The figure is a plot generated fromcapillary electrophoresis of products of multiplex amplification of DNAfrom paired normal and colon tumor samples, using the same nine locusMSI multiplex and labeled primers used in FIG. 15.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The following definitions are intended to assist in providing a clearand consistent understanding of the scope and detail of the followingterms, as used to describe and define the present invention:

“Allele”, as used herein, refers to one of several alternative forms ofa gene or DNA sequence at a specific chromosomal location (locus). Ateach autosomal locus an individual possesses two alleles, one inheritedfrom the father and one from the mother.

“Amplify”, as used herein, refers to a process whereby multiple copiesare made of one particular locus of a nucleic acid, such as genomic DNA.Amplification can be accomplished using any one of a number of knownmeans, including but not limited to the polymerase chain reaction (PCR)(Saiki, R. K., et al., 1985 Science 230: 1350-1354), transcription basedamplification (Kwoh, D. Y., and Kwoh, T. J., American BiotechnologyLaboratory, October, 1990) and strand displacement amplification (SDA)(Walker, G. T., et al., 1992 Proc. Natl. Acad. Sci., U.S.A. 89:392-396).

“Co-amplify”, as used herein, refers to a process whereby multiplecopies are made of two or more loci in the same container, in a singleamplification reaction.

“DNA polymorphism”, as used herein, refers to the existence of two ormore alleles for a given locus in the population. “Locus” or “geneticlocus”, as used herein, refers to a unique chromosomal location definingthe position of an individual gene or DNA sequence. “Locus-specificprimer”, as used herein, refers to a primer that specifically hybridizeswith a portion of the stated locus or its complementary strand, at leastfor one allele of the locus, and does not hybridize efficiently withother DNA sequences under the conditions used in the amplificationmethod.

“Loss of Heterozygosity” (hereinafter, “LOH”), as used herein, refers tothe loss of alleles on one chromosome detected by assaying for markersfor which an individual is constitutionally heterozygous. Specifically,LOH can be observed upon amplification of two different samples ofgenomic DNA from a particular subject, one sample originating fromnormal biological material and the other originating from a tumor orpre-cancerous tissues. The tumor exhibits LOH if DNA from the normalbiological material produces amplified alleles of two different lengthsand the tumor samples produces only one of the two lengths of amplifiedalleles at the same locus.

“Microsatellite Locus”, as used herein, refers to a region of genomicDNA that contains short, repetitive sequence elements of one (1) toseven (7), more preferably one (1) to five (5), most preferably one (1)to four (4) base pairs in length. Each sequence repeated at least oncewithin a microsatellite locus is referred to herein as a “repeat unit.”Each microsatellite locus preferably includes at least seven repeatunits, more preferably at least ten repeat units, and most preferably atleast twenty repeat units.

“Microsatellite Instability” (hereinafter, “MSI”), as used herein,refers to a form of genetic instability in which alleles of genomic DNAobtained from certain tissue, cells, or bodily fluids of a given subjectchange in length at a microsatellite locus. Specifically, MSI can beobserved upon amplification of two different samples of genomic DNA froma particular subject, such as DNA from healthy and cancerous tissue,wherein the normal sample produces amplified alleles of one or twodifferent lengths and the tumor sample produces amplified alleleswherein at least one of the alleles is of a different length from theamplified alleles of the normal sample of DNA at that locus. MSIgenerally appears as a result of the insertion or deletion of at leastone repeat unit at a microsatellite locus.

“MSI-H”, as used herein, is a term used to classify tumors as having ahigh frequency of MSI. When five microsatellite loci are analyzed, suchas the five microsatellite loci of selected by a workshop on HNPCC atthe National Cancer Institute in 1998 for use in the detection of HNPCC,a tumor is classified as MSI-H when at least two of the loci showinstability (Boland, 1998 Cancer Research 58: 5248-5257). When more thanfive microsatellite loci are analyzed, a tumor is classified as MSI-Hwhen at least 30% of the microsatellite loci of genomic DNA originatingfrom the tumor is are found to be unstable.

“MSI-L”, as used herein, is a term used to classify tumors as having alow frequency of MSI. When five microsatellite loci are analyzed, suchas the five microsatellite loci of selected by a workshop on HNPCC atthe National Cancer Institute in 1998 for use in the detection of HNPCC,a tumor is classified as MSI-L when only one of the loci showsinstability. When more than five microsatellite loci are analyzed, atumor is classified as MSI-L when less than 30% of the microsatelliteloci of genomic DNA originating from the tumor is are found to beunstable. MSI-L tumors are thought to represent a distinct mutatorphenotype with potentially different molecular etiology than MSI-Htumors (Thibodeau, 1998; Wu et al., 1999, Am J Hum Genetics65:1291-1298). To accurately distinguish MSI-H and MSI-L phenotypes ithas been recommended that more than five microsatellite markers beanalyzed (Boland, 1998, supra; Frazer et al., 1999 Oncology Research6:497-505).

“MSS”, as used herein, refers to tumors which are microsatellite stable,when no microsatellite loci exhibit instability. The distinction betweenMSI-L and MSS can also only be accomplished when a significantly greaternumber of markers than five are utilized. The National Cancer Instituterecommended use of an additional 19 mono- and dinucleotide repeat locifor this purpose, and for the purpose of making clearer distinctionsbetween MSI-H and MSI-L tumors, as described above (Boland, 1998,supra).

“MSI-L/S”, as used herein, refers to all classified as either MSI-L orMSS.

“Microsatellite marker”, as used herein, refers to a fragment of genomicDNA which includes a microsatellite repeat and nucleic acid sequencesflanking the repeat region.

“Monomorphic”, as used herein, refers to a locus of genomic DNA whereonly one allele pattern has been found to be present in the normalgenomic DNA of all members of a population.

“Nucleotide”, as used herein, refers to a basic unit of a DNA molecule,which includes one unit of a phosphatidyl back bone and one of fourbases, adenine (“A”); thymine (“T”); guanine (“G”); and cytosine (“C”).

“Polymerase chain reaction” or “PCR”, as used herein, refers to atechnique in which cycles of denaturation, annealing with primer, andextension with DNA polymerase are used to amplify the number of copiesof a target DNA sequence by approximately 10⁶ times or more. Thepolymerase chain reaction process for amplifying nucleic acid is coveredby U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated hereinby reference for a description of the process.

“Primer”, as used herein, refers to a single-stranded oligonucleotide orDNA fragment which hybridizes with a strand of a locus of target DNA insuch a manner that the 3′ terminus of the primer may act as a site ofpolymerization using a DNA polymerase enzyme.

“Primer pair”, as used herein, refers to a pair of primers whichhybridize to opposite strands a target DNA molecule, to regions of thetarget DNA which flank a nucleotide sequence to be amplified.

“Primer site”, as used herein, refers to the area of the target DNA towhich a primer hybridizes.

“Quasi-monomorphic”, as used herein, refers to a locus of genomic DNAwhere only one allele pattern has been found to be present in the normalgenomic DNA of almost all the members of a population.

“Stutter”, as used herein, refers to a minor fragment observed afteramplification of a microsatellite locus, one or more repeat unit lengthssmaller than the predominant fragment or allele. It is believed toresult from a DNA polymerase slippage event during the amplificationprocess (Levinson & Gutman, 1987 Molecular Biology Evolution 4:203;Schlotterer and Tautz, 1992 Nucleic Acids Research 20:211).

B. Selection of Loci to be Amplified or Co-Amplified

At least one MSI locus amplified or co-amplified in each of theembodiments of the present invention illustrated and discussed herein isa mononucleotide repeat locus. Such loci have been shown to be verysusceptible to alteration in tumors with dysfunctional DNA mismatchrepair systems (Parsons, 1995 supra), making such loci particularlyuseful for the detection of cancer and other diseases associated withdysfunctional DNA mismatch repair systems. One group of researchersreported that by amplifying and analyzing a single mononucleotide repeatlocus, BAT-26, they were able to correctly confirm the MSI-H status of159 out of 160 (99.4% accuracy) tumor samples (Hoang et al., 1997 CancerResearch 57:300-303).

Some mononucleotide repeat loci, including BAT-26, have also beenidentified as having quasi-monomorphic properties. Monomorphic orquasi-monomorphic properties make the comparison of normal/tumor pairssimpler, since PCR products from normal samples are generally all thesame size and any alterations in tumor samples are easily identified.

The principal draw-back to using a mononucleotide repeat locus in theanalysis of genomic DNA is that amplification of any such locus resultsin a large number of extraneous amplified fragments of DNA of variouslengths, the product of “stutter” during the amplification reaction.Such artifacts are present to a lesser degree in the products ofamplifying loci with increasingly longer repeat units. For anillustration of the relationship between repeat unit length and thepresence of extraneous amplified fragments, see FIG. 2. FIG. 2 showsincreased stutter artifacts with decreasing repeat unit length frompentanucleotide to mononucleotide repeat loci.

When a mononucleotide locus is monomorphic or quasi-monomorphic,however, one can readily detect shifts in the size of an allele,indicating MSI, even in the presence of a high degree of stutter. When alocus is quasi-monomorphic, detection of shifts in size can be done bycomparison of amplified alleles from genomic DNA from biologicalmaterial of an individual, such as tumor tissue or bodily fluids,suspected of exhibiting microsatellite instability to the most commonlyobserved allele size at that locus in a population. This feature enablesone to use a single standard or panel of standard allele patterns toanalyze individual results, minimizing the amount of samples which mustbe taken from an individual in order to detect microsatelliteinstability in certain genomic DNA of the individual.

At least one of the microsatellite loci amplified in the method or usingthe kit of the present invention is preferably a mononucleotide repeatlocus, more preferably a quasi-monomorphic mononucleotide repeat locus.The mononucleotide repeat locus selected for use in the methods and kitsof the present invention is preferably unstable in cancerous biologicalmaterial, but not in normal biological material. BAT-25, BAT-26 andBAT-40 have been identified as mononucleotide repeat loci useful in theidentification of MSI in colorectal tumors characteristic of HereditaryNonpolyposis Colon Cancer (Zhou et al., 1998 Genes, Chromosomes & Cancer21:101-107; Boland et al, 1998 Cancer Research 58:5248-5257, Dietmaieret al., 1997 Cancer Research 57:4749-4756; Hoang et al., 1997 CancerResearch 57:300-303). Two additional loci, identified herein as MONO-11and MONO-15 were identified through a search of a public computerizeddatabase of sequence information (GenBank), and found to have thepreferred characteristics for such loci, identified above. The searchfor and identification of mononucleotide repeat loci suitable for use inthe present invention is illustrated in Example 2. Similar techniquescould be used to identify other mononucleotide repeat loci suitable foruse in the methods and kits of the present invention.

The mononucleotide repeat loci amplified or co-amplified according tothe present methods or using the present kits are preferablyquasi-monomorphic and exhibit instability in the type of tissue ofinterest for a given application. MONO-11 and MONO-15, BAT-25, andparticularly BAT-26 are all quasi-monomorphic monomorphic and exhibitinstability in several cancerous tumor tissues. All fourquasi-monomorphic mononucleotide repeat loci have been found to beparticularly useful in the methods and kits of the present invention.BAT-40 has also been found to be useful in the methods and kits of thepresent invention, due to its instability in several cancerous tumortissues. However, BAT-40 is not a quasi-monomorphic locus. At least one,more preferably at least two mononucleotide repeat microsatellite lociare amplified or co-amplified in the method of the present invention.

At least one mononucleotide repeat locus and at least twotetranucleotide repeat loci are co-amplified and analyzed according toat least some embodiments of the method and kits of the presentinvention. Tetranucleotide repeat loci inherently generate very fewstutter artifacts when amplified, compared to microsatellite loci withshorter repeat units, particularly compared to mono- and dinucleotiderepeat loci. (See, e.g., FIG. 2.) Such artifacts can be difficult todistinguish from MSI if a shifted allele occurs at the stutter positionof the second allele. Therefore, concerns about interpretation, and theneed for quasi-monomorphism in order to make data interpretationpossible is not present, as it is for mononucleotide repeat loci. Infact, one can even use tetranucleotide repeat loci which are highlypolymorphic in a population, provided it is stable within an individualsubject. Such loci are commonly used in DNA typing.

As with any locus to be amplified in any method or using any kit of thepresent invention, the tetranucleotide repeat loci are preferablyselected on the basis of being stable in the DNA of an individual exceptin the type of biological material of interest. Preferredtetranucleotide repeat loci used in the methods and kits of the presentinvention include: FGA, D1S518, D1S547, D1S1677, D2S1790, D3S2432,D5S818, D5S2849, D6S1053, D7S3046, D7S1808, D7S3070, D8S1179, D9S2169,D10S1426, D10S2470, D12S391, D17S1294, D17S1299, and D18S51.

Additional mononucleotide or tetranucleotide loci with the samepreferred criteria described above are preferably co-amplified with theset of at least three microsatellite loci described above. However, itis contemplated that microsatellite loci other than mononucleotiderepeat or tetranucleotide repeat loci could be included in the set of atleast three microsatellite loci co-amplified and analyzed according tothe method or using the kit of the present invention.

Preferred methods for selection of loci and sets of loci amplified andanalyzed according to the methods or using the kits of the presentinvention are discussed further, herein below. However, once the methodand materials of this invention are disclosed, additional methods ofselecting loci, primer pairs, and amplification techniques for use inthe method and kit of this invention are likely to be suggested to oneskilled in the art. All such methods are intended to be within the scopeof the appended claims.

C. Additional Screening of Loci

When the method or kit of the present invention is to be used inclinical diagnostic assays to be used to determine treatment andprognosis of disease, it must be designed to produce results which arehighly accurate (low false negatives) and specific (low false positiverate). Informative microsatellite loci are preferably identified byscreening, more preferably by very extensive screening (see Examples 1and 2). However, even the most informative microsatellite loci are not100% sensitive and 100% specific.

The power of individual markers at detecting the presence of MSI intissue associated with a particular disease, such as cancerous tumors,can be increased tremendously by multiplexing multiple markers.Increased information yielded from amplifying and analyzing greaternumbers of loci results in increased confidence and accuracy ininterpreting test results. To obtain needed sensitivity in detecting ordiagnosing diseases such as cancer, it has been recommended that oneanalyze five or more highly informative microsatellite loci (Boland,1998 Cancer Research 58: 5248-5257). Multiplexing of microsatellite locifurther simplifies MSI analysis by allowing simultaneous amplificationand analysis of all multiple loci, while reducing the amount ofoften-limited DNA required for amplification.

Another common problem in MSI determination relates to the occurrence ofan intermediate MSI phenotype where only a small percentage (<30%) ofmicrosatellite markers are altered in tumors (Boland, 1998, supra).These MSI-low tumors are thought to represent a distinct mutatorphenotype with potentially different molecular etiology than MSI-Htumors (Thibodeau et al., 1993 Science 260: 816-8; Wu et al., 1999 Am JHum Genetics 65:1291-1298; Kolodner et al., 1999 Cancer Research59:5068-5074; Wijnen et al., 1999 Nature Genetics 23:142-144). It is notclear however if there is a real difference between MSI-L and MSStumors. For purposes of diagnosis, MSI-L and MSS tumors are generallyconsidered as one stable phenotypic class. To accurately distinguishMSI-H and MSI-L phenotypes it has been recommended that multiplemicrosatellite markers be analyzed (Boland, 1998; Frazer, 1999 supra).

It is contemplated that when the loci are to be co-amplified andanalyzed in a multiplex amplification reaction, additional factors aretaken into account, including ease and accuracy of interpretation ofdata. One of the primary factors affecting accurate data interpretationis the amount of stutter that occurs at microsatellite loci during PCR.Tetranucleotide repeat loci were chosen for inclusion in the MSImultiplex analyzed according to the method and using the kit of thepresent invention because they display considerably less stutter thatshorter repeat types like dinucleotides (FIG. 2). However, carefulselection of loci is still necessary in choosing low stutter locibecause % stutter can vary considerably even within a particular repeattype (FIG. 4). Mononucleotide repeat loci were chosen for individualanalysis and for inclusion in the MSI Multiplex because of high rates ofinstability in diseased biological material of interest.

Incidence of LOH is another factor in the selection of MSI loci to beamplified and analyzed in the methods or kits of the present invention.LOH can result in misidentification of a missing normal allele at amicrosatellite marker as an indication of MSI when no other novelfragments are present for that marker. Specifically, one cannot easilydiscern whether this represents true LOH or MSI in which the shiftedallele has co-migrated with the remaining wild-type allele. In order tominimize the problem described above, the microsatellite markersselected for use in the present methods and kits preferably exhibit alow frequency of LOH, preferably no more than about 20% LOH, morepreferably no more than about 14% LOH, even more preferably, no morethan about 3% LOH.

It is a relatively uncommon occurrence for a microsatellite marker topossess all necessary attributes described above (i.e., highsensitivity, high specificity, low stutter, low LOH). The threshold foran MSI analysis system to be used in a diagnostic test is even higher,requiring robust and reproducible results from multiple loci in oneassay using small quantities of DNA from difficult samples and be ableto distinguish between MSI-L and MSI-H phenotypes. All the specificpreferred mono- and tetranucleotide repeat loci identified herein aboveas being preferred for use in the present invention were found to meeteach of the criteria for MSI loci suitable for use in diagnosticanalysis, set forth herein above.

Additional loci selection criteria particular to the two principal typesof MSI loci amplified in the preferred multiplex analysis methods andusing the kits of the present loci are described below.

D. Design of Primers

Primers for one or more microsatellite loci are provided in eachembodiment of the method and kit of the present invention. At least oneprimer is provided for each locus, more preferably at least two primersfor each locus, with at least two primers being in the form of a primerpair which flanks the locus. When the primers are to be used in amultiplex amplification reaction it is preferable to select primers andamplification conditions which generate amplified alleles from multipleco-amplified loci which do not overlap in size or, if they do overlap insize, are labeled in a way which enables one to differentiate betweenthe overlapping alleles.

Primers suitable for the amplification of individual loci preferablyco-amplified according to the methods of the present invention areprovided in Example 4, Table 9, herein below. Primers suitable for usein one preferred multiplex of nine loci (i.e., BAT-25, D10S1426,D3S2432, BAT-26, D7S3046, D7S3070, MONO-15, D1S518, and D7S1808) aredescribed in Example 6, Table 11. Primers suitable for use in a morepreferred multiplex of nine loci (i.e., BAT-25, D10S1426, D3S2432,BAT-26, D7S3046, D7S3070, MONO-15, BAT-40, and D7S1808) are described inExample 8, Table 13. Guidance for designing these and other multiplexesis provided, below. It is contemplated that other primers suitable foramplifying the same loci or other sets of loci falling within the scopeof the present invention could be determined by one of ordinary skill inthe art.

E. Design and Testing of MSI Multiplex

The method of multiplex analysis of microsatellite loci of the presentinvention contemplates selecting an appropriate set of loci, primers,and amplification protocols to generate amplified alleles from multipleco-amplified loci which preferably do not overlap in size or, morepreferably, which are labeled in a way which enables one todifferentiate between the alleles from different loci which overlap insize. Combinations of loci may be rejected for either of the above tworeasons, or because, in combination, one or more of the loci do notproduce adequate product yield, or fragments which do not representauthentic alleles are produced in this reaction.

The following factors are preferably taken into consideration indeciding upon which loci to include in a multiplex of the presentinvention. To effectively design the microsatellite multiplex, sizeranges for alleles at each locus are determined. This information isused to facilitate separation of alleles between all the different loci,since any overlap could result in an allele from one locus beinginappropriately identified as instability at another locus.

The amount of stutter exhibited by non-mononucleotide repeat loci isalso preferably taken into consideration; as the amount of stutterexhibited by a locus can be a major factor in the ease and accuracy ofinterpretation of data. It is preferable to conduct a population studyto determine the level of stutter present for each non-mononucleotiderepeat locus. As noted above, tetranucleotide repeat markers displayconsiderably less stutter that shorter repeat types like dinucleotidesand therefore can be accurately scored in MSI assays (FIGS. 2 and3)(Bacher & Schumm, 1998 Profiles in DNA 2(2):3-6). Note that evenwithin a class of microsatellite loci, such as tetra- andpentanucleotide repeat loci, known to exhibit low stutter, the percentstutter can vary considerably within the repeat type (FIG. 3; see alsoFIG. 2) (Micka et al., 1999, supra).

Although at least one mononucleotide and at least two tetranucleotiderepeat loci are included in the multiplex of MSI loci co-amplifiedaccording to the method or using the kit of the present invention,additional mononucleotide and/or tetranucleotide repeat loci can beincluded in the multiplex. It is also contemplated that multisatelliteloci other than mono- or tetranucleotide repeat loci meeting the same orsimilar criteria to the criteria described above would be included inthe multiplex.

The multiplex analyzed according to the present invention preferablyincludes a set of at least three MSI loci. It more preferably includes aset of at least five MSI loci, even more preferably a set of at leastnine MSI loci. When the multiplex is a set of at least nine loci, it ispreferably a set of at least the following loci: BAT-25, D10S1426,D3S2432, BAT-26, D7S3046, D7S3070, MONO-15, D1S518, and D7S1808, or morepreferably a set of at least the following loci: BAT-25, D10S1426,D3S2432, BAT-26, D7S3046, D7S3070, BAT-40, MONO-15 and D7S1808. A listof primers suitable for use in the first multiplex is provided in Table11 of Example 6, below. A list of primers suitable for use in thesecond, more preferred multiplex is provided in Table 13 of Example 8,below.

It is also contemplated that other factors, such as successfulcombinations of materials and methods, are taken into consideration indesigning a multiplex of MSI loci. Determination of such additionalfactors can be determined by following the selection methods andguidelines disclosed herein, and by using techniques known to one ofordinary skill in the art of the present invention. Specifically, thesame or substantially similar techniques can be used to identify thepreferred MSI loci and sets of MSI loci described herein below to selectprimer pair sequences, and to adjust primer concentrations to identifyan equilibrium in which all included loci may be amplified. In otherwords, once the method and materials of this invention are disclosed,various methods of selecting loci, primer pairs, and amplificationtechniques for use in the method and kit of this invention are likely tobe suggested to one skilled in the art. All such methods are intended tobe within the scope of the present claims.

F. Sources of Genomic DNA

The genomic DNA amplified or co-amplified according to the methods ofthe present invention originates from biological material from anindividual subject, preferably a mammal, more preferably from a dog,cat, horse, sheep, mouse, rat, rabbit, monkey, or human, even morepreferably from a human or a mouse, and most preferably from a humanbeing. The biological material can be any tissue, cells, or biologicalfluid from the subject which contains genomic DNA. The biologicalmaterial is preferably selected from the group consisting of tumortissue, disseminated cells, feces, blood cells, blood plasma, serum,lymph nodes, urine, and other bodily fluids.

The biological material can be in the form of tissue samples fixed informalin and embedded in paraffin (hereinafter “PET”). Tissue samplesfrom biopsies are commonly stored in PET for long term preservation.Formalin creates cross-linkages within the tissue sample which can bedifficult to break, sometimes resulting in low DNA yields. Anotherproblem associated with formalin-fixed paraffin-embedded samples isamplification of longer fragments is often problematic. When DNA fromsuch samples is used in multiplex amplification reactions, a significantdecrease in peak heights is seen with increasing fragment size. Themicrosatellite analysis method and kit of the present invention arepreferably designed to amplify and analyze DNA from PET tissue samples.(See Example 7 for an illustration of amplification of such samplesusing a method of the present invention.).

When the method or kit of the present invention is used in the analysisor detection of tumors, at least one sample of genomic DNA analyzedoriginates from a tumor. When a monomorphic or quasi-monomorphic locus,such as MONO-11 or MONO-15 is amplified, the size of the resultingamplified alleles can be compared to the most commonly observed allelesize at that locus in the general population. The present method and kitis preferably used to diagnose or detect tumors by co-amplifying atleast two different samples of DNA from the same individual, wherein oneof the two samples originates from normal non-cancerous biologicalmaterial.

The present invention is further explained by the following exampleswhich should not be construed by way of limiting the scope of thepresent invention.

EXAMPLE 1 Screening Microsatellite Markers for Frequency of MSI

In this example, microsatellite markers in DNA isolated from tumors werecompared to microsatellite markers in DNA isolated from normal tissue orcells in order to detect MSI. Specifically, microsatellite loci wereamplified from paired normal/tumor DNA samples and genotyped. If one ormore different alleles were present in the tumor DNA sample that werenot found in normal sample from the same individual, then it was scoredas MSI positive. Dinucleotide, tetranucleotide and pentanucleotiderepeat microsatellite markers were analyzed for frequency of alterationto determine the relative sensitivity of particular markers to MSI.Detailed information about the specific procedures used in this exampleare provided herein, below.

Tissues and DNA isolation. Matched normal (blood) and neoplastic tissuesamples for 39 patients were obtained from the Cooperative Human TissueNetwork (hereinafter, “CHTN”)(Ohio State University, Columbus, Ohio).After surgical resection, tissue samples were frozen in liquid nitrogenand stored at −70° C. Blood samples were collected by venipuncture usingvacuum tubes. DNA extraction from blood and solid tissues was performedeither by standard Phenol/chloroform method (Sambrook et al., 1989Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Press, ColdSprings Harbor, N.Y.) or with QIAamp Blood and Tissue Kit (QIAGEN, SantaClarita, Calif.) following manufacturer's protocol.

PCR and Microsatellite Analysis. Fluorescently labeled primers from 275microsatellite loci were used to amplify template DNA from normal/tumorpairs of samples. Two hundred and forty-five tetranucleotide repeatmarkers from the Research Genetics CHLC/Weber Human Screening SetVersion 9.0 were evaluated (Research Genetics, Huntsville, Ala.).Additional primer sets for tetranucleotide and pentanucleotide repeatmarkers were obtained from Promega Corporation (Madison, Wis.)(PowerPlex™ 16 System contains D3S1358, TH01, D21S11, D18S51, Penta E,D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX,and FGA loci). Pentanucleotide repeat markers TP53, Penta A, Penta B,Penta C, Penta D, Penta E, Penta F and Penta G or were customsynthesized (Promega Corporation, Madison, Wis.) using sequence datafrom public databases Dinucleotide markers included for comparisonpurposes (D8S254, NM23, D18S35, D5S346, TP53-di, D2S123, D1S2883,D3S1611, D7S501) were obtained from PE Biosystems (now doing business asApplied Biosystems Group, Foster City, Calif.).

Markers from Research Genetics, Human Screening Set Version 9.0, weremultiplexed and screened for MSI using 2.5 ng of DNA in 10 μl PCRreactions described below. Other loci were evaluated as monoplexes using1 ng DNA in 25 μl PCR reactions as described below. All markers were PCRamplified under the same conditions in using a Perkin-Elmer® GeneAmp PCRSystem 9600 Thermal Cycler, except as indicated otherwise below.Microsatellite markers from the PowerPlex™ 16 System (Technical Manual#TMD012, Promega Corporation, Madison, Wis.) and dinucleotide repeatmarkers from the Microsatellite RER Assay system (see product literaturefrom PE Biosystems, non Applied Biosystems, Foster City, Calif.) wereanalyzed following manufacturer's protocol.

TABLE 1 10 μl triplex PCR reaction for Research Genetics markers VolumePer PCR Master Mix Component Sample Nuclease Free Water 3.30 μl 10×GoldSTHR Buffer (Promega) 1.00 μl Primer 1 0.50 μl Primer 2 0.50 μlPrimer 3 0.50 μl Primer 4 0.50 μl Primer 5 0.50 μl Primer 6 0.50 μlAmpliTaq Gold DNA Polymerase (5 Units/μl) 0.15 μl (Perkin Elmer) DNA (1ng/μl) 2.50 μl Total Reaction Volume 10.00 μl

TABLE 2 25 μl PCR reaction Volume Per PCR Master Mix Component SampleNuclease Free Water 17.45 μl GoldSTHR 10× Buffer (Promega) 2.50 μl 10×Primer Pair Mix (10 μM) 2.50 μl AmpliTaq Gold DNA Polymerase (5Units/μl) 0.05 μl (Perkin Elmer) Template DNA (0.4 ng/μl) 2.50 μl TotalReaction Volume 25.00 μl

TABLE 3 Cycling profile for PE 9600 Thermal Cycler  1 cycle 95° C. for11 minutes  1 cycle 96° C. for 1 minute 10 cycles 94° C. for 30 secondsramp 68 seconds to 56° C., hold for 30 seconds ramp 50 seconds to 70°C., hold for 45 seconds 20 cycles 94° C. for 30 seconds ramp 60 secondsto 56° C., hold for 30 seconds ramp 50 seconds to 70° C., hold for 45seconds  1 cycle 60° C. for 30 minutes  1 cycle Soak 4° C.

One microliter of PCR product (Research Genetics markers were firstdiluted 1:4 in 1× GoldSTHR PCR buffer) was combined with 1 μl ofInternal Lane Standard (Promega Corporation, Madison, Wis.) and 24 μldeionized formamide. Samples were denatured by heating at 95° C. for 3minutes and immediately chilled on ice for 3 minutes. Separation anddetection of amplified fragments was performed on an ABI PRISM® 310Genetic Analyzer following the standard protocol recommended in theUser's Manual with the following settings: 5 second Injection Time, 15kV Injection Voltage, 15 kV Run Voltage, 60° C. Run Temperature, and 28minute Run Time.

Assay Interpretation. Identification of normal and tumor allele sizeswas accomplished by examining the appropriate electropherogram from theABI PRISM 310 Genetic Analyzer (Applied Biosystems) and determining thepredominant peaks for each locus. One or two peaks or alleles can bepresent for each locus in normal samples depending upon whetherindividual is homozygous or heterozygous for a particular marker.Allelic patterns or genotypes for normal and tumor pairs were comparedand scored as MSI positive if one or more different alleles were presentin the tumor DNA samples that were not found in normal sample from thesame individual.

A wide range in frequency of alteration was observed in between samplesand between markers evaluated. Samples were divided into two groupsbased on the frequency of alteration using guidelines recommended in NCIWorkshop on MSI (Boland et al., 1998). Samples with greater that 30-40%of markers exhibiting alteration in tumor samples were classified asMSI-H and <30-40% as MSI-L. Samples with no alterations were classifiedas microsatellite stable (MSS). Based on this definition of MSIphenotypes, nine samples were classified as MSI-H and the remaining 30as either MSI-L or MSS.

The tetra- and pentanucleotide repeat loci exhibited the smallest amountof stutter of the loci screened, above. See FIG. 4 for a plot of the %stutter results observed at the tetra- and pentanucleotide repeat loci.The tetranucleotide repeat markers also varied in frequency ofalteration, ranging from 0 to 100% MSI in the MSI-H group (FIG. 5).Pentanucleotide markers, in general, displayed low levels of MSI (FIG.6). Microsatellite markers showing high sensitivity to MSI (>88% MSIwith MSI-H samples) and high specificity (<8% MSI with MSI-L and MSSsamples) with the CHTN samples were selected for further evaluation with20 additional normal/tumor colon cancer samples from Mayo Clinic(Rochester, Minn.) (see Example 5).

EXAMPLE 2 Identification and Characterization of Mononucleotide RepeatLoci

Due to the highly informative nature of mononucleotide repeat loci indetermining MSI phenotype, we also investigated poly (A) regions of thehuman genome as a new source of markers for MSI assays. To accomplishthis, mononucleotide repeats were identified from GenBank using BLASTN(Altschul, et al. 1990 J. Mol. Biol. 215:402-410) searches for(A)₃₀(N)₃₀ sequences. The (N)₃₀ sequence was added to eliminate frequentmRNA hits and to assure that flanking sequence was available fordesigning primers for PCR. Next, flanking primers were designed for 33GenBank DNA sequences using Oligo Primer Analysis Software version 6.0(National Biosciences, Inc., Plymouth, Minn.) to amplify the regioncontaining the poly (A) repeat. Evaluation of loci was performed using 9MSI-H and 30 MSS colon cancer samples and corresponding normal DNAsamples. Protocols for PCR, detection and analysis are described inExample 1.

Two characteristics were screened for in the new loci. First, loci werescreened for which could detect MSI in the MSI-H group and not in theMSS group. Secondly, loci were selected on the basis of beingmonomorphic or nearly monomorphic (quasi-monomorphic). The monomorphicnature of the new loci was determined by genotyping 96 samples from 5racial groups (African-American, Asian-American, Caucasian-American,Hispanic-American, Indian-American). Screening of 33 mononucleotiderepeat loci revealed four new mononucleotide repeat loci (MONO-3,MONO-11, MONO-15, and MONO-19) that displayed high sensitivity to MSI(Table 4 and FIG. 7) and were relatively homozygous and monomorphic(Table 5). The degree of homozygosity and mono-morphism detected at eachsuch locus is shown on Table 5.

TABLE 4 Results from Screening of Mononucleotide Repeat Loci MSI MONO-MONO- MONO- Type BAT-25 BAT-26 MONO-3 11 15 19 MSI-H 100% 100% 100% 100%100% 100% MSI-L or 0% 0% 0% 0% 0% 0% MSS

TABLE 5 Polymorphism Level of Mononucleotide Repeat Loci BAT-25 BAT-26MONO-11 MONO-15 % Homo- 95% (82/86) 95% (89/94) 89% (76/85) 99% (87/88)zygosity % Mono- 95% 95% 89% 99% morphic

EXAMPLE 3 Population Studies

A population study was conducted in which 93 samples fromAfrican-American individuals were genotyped using preferredmicrosatellite loci selected as candidates for multiplexing in thestudies illustrated in Examples 1 and 2, above. See Table 6, below, andTable 3, above, for the amplification conditions used. See Table 7,below, for a list of the loci amplified and analyzed in this study. Inaddition, a pooled Human Diversity DNA sample and control CEPH DNAs1331-1 and 1331-2 (Coriell Cell Repository, Camden, N.J.) were includedin the screening population. African-American samples were used becausethey contain the greatest genetic diversity found in all racial groups.

To facilitate screening of 96 samples with 22 different microsatellitemarkers, selected markers were multiplexed in small groups of three.Multiplexed primer sets were used to amplify individual sample DNAsusing conditions described below.

TABLE 6 25 μl PCR reaction Volume Per PCR Master Mix Component SampleNuclease Free Water 17.30 μl GoldSTHR 10× Buffer (Promega) 2.50 μl 10×Triplex Primer Mix (1 to 10 μM each) 2.50 μl AmpliTaq Gold DNAPolymerase (5 Units/μl) 0.20 μl (Perkin Elmer) Template DNA (0.4 ng/μl)2.50 μl Total Reaction Volume 25.00 μl

The results of the population study are summarized in Table 7. The sizeof the smallest and largest allele for each locus was identified todetermine allele size range.

To calculate percent stutter, the peak height of the stutter band wasdivided by the peak height generated by the true allele, then multipliedby 100. Minimum and maximum stutter values were calculated for eachlocus as well as the combined average percent stutter from 20 randomsamples.

TABLE 7 Summary of Results of Population Study Allele Size Range AverageLocus GenBank ID # GenBank Pop Study % Stutter BAT-25 U63834  18 bp 42bp ND BAT-26 U41210  18 bp 12 bp ND MONO-11 AC007684 ND 14 bp ND MONO-15AC007684 ND  6 bp ND D1S547 G07828  46 bp 26 bp 4.9 D1S518 G07854  48 bpND ND D1S1677 G09926  40 bp 35 bp 9.7 D2S1790 G08190  68 bp 44 bp 7.8D3S2432 G08240  67 bp 40 bp 8.0 D5S818 G08446  36 bp ND ND D5S2849G15752  40 bp 37 bp 5.5 D6S1053 G08556  48 bp 36 bp 6.9 D7S1808 G08643 58 bp 44 bp 7.6 D7S3046 G10353  48 bp 71 bp 12.9 D7S3070 G27340  44 bp44 bp 10.3 D8S1179 G08710  44 bp ND ND D9S2169 G08748  12 bp ND NDD10S677 G12433  28 bp 40 bp 5.5 D10S1426 G08812  28 bp ND ND D10S2470G10285  48 bp 29 bp 5.9 D12S391 G08921  52 bp 48 bp 7.6 D17S1294 G07967 44 bp 28 bp 7.2 D17S1299 G07952  40 bp ND ND D18S51 L18333  76 bp ND NDFGA M64982 120 bp ND ND

EXAMPLE 4 MSI Multiplex Design

In order to develop a multiplex MSI assay system which is highlysensitive to MSI, with minimal stutter, and minimal incidence of LOH,the criteria listed in Table 8, below, were used to screen lociidentified in the Examples above as possible candidates for use in MSIanalysis:

TABLE 8 MSI Loci Specifications for Use in Multiplex Monoplexspecifications Tetranucleotides >70% MSI in MSI-H samples <8% MSI withMSI-L and MSS samples LOH <14% in MSI-H samples Average % Stutter <13%Mononucleotides 100% MSI in MSI-H samples 0% MSI with MSI-L and MSSsamples Multiplex specifications 9 loci; 3 mono- and 6 tetranucleotidesAll amplicons <250 bp Robust amplification of DNA from PET samplesRobust amplification of 1 to 2 ng DNA Balanced peak heights between allloci in multiplex Sensitivity >99.9% Specificity >99.9%The loci listed in Table 9, below, were identified as loci meeting thespecifications listed in Table 8, above.

TABLE 9 Preferred Microsatellite Loci for Multiplexing Re- GenBankPrimer % MSI % MSI peat Accession SEQ. (MSI- % LOH (MSS or Locus TypeNo. ID. H) (MSI-H) MSI-L) BAT-25 Mono U63834 1, 2 100% 0% 0% BAT-26 MonoU41210 3, 4 100% 0% 0% MONO-11 Mono AC007684 5, 6 100% 0% 0% MONO-15Mono AC007684 7, 8 100% 0% 0% D1S518 Tetra G07854  9, 10 83% 0% 0%D1S547 Tetra G07828 11, 12 78% 3% 0% D1S1677 Tetra G09926 13, 14 80% 0%3% D2S1790 Tetra G08190 15, 16 82% 3% 3% D3S2432 Tetra G08240 17, 18 83%3% 3% D5S818 Tetra G08446 19, 20 72% 14% 3% D5S2849 Tetra G15752 21, 2276% 7% 0% D6S1053 Tetra G08556 23, 24 76% 0% 0% D7S1808 Tetra G08643 25,26 90% 0% 0% D7S3046 Tetra G10353 27, 28 93% 0% 0% D7S3070 Tetra G2734029, 30 86% 3% 3% D8S1179 Tetra G08710 31, 32 75% 7% 7% D9S2169 TetraG08748 33, 34 72% 3% 0% D10S1426 Tetra G08812 35, 36 86% 3% 0% D10S2470Tetra G10285 37, 38 83% 3% 0% D12S391 Tetra G08921 39, 40 79% 3% 0%D17S1294 Tetra G07967 41, 42 86% 3% 0% D17S1299 Tetra G07952 43, 44 79%3% 0% D18S51 Tetra L18333 45, 46 75% 7% 0% FGA Tetra M64982 47, 48 82%7% 7% * MSI-H samples: N = 29 and MSI-L/S samples N = 30.

EXAMPLE 5 Analysis of Mismatch Repair Genes

In order to determine the underlying cause of MSI in MSI-H tumor samplesused in developing the Multiplex MSI Assay, protein expression levelsfor MLH1 and MSH2 genes were examined. Immunohistochemical analysis ofparaffin-embedded tissues from eight MSI-H samples was performed asdescribed in Thibodeau et al. (Cancer Research 58, 1713-1718). Lack ofprotein expression in MLH1 and MSH2 genes is expected in tumor samplesexhibiting high levels of MSI and is an indication of dysfunctionalmismatch repair system.

The results of the immunohistochemical assays on the MSI-H tumor samplesis shown in Table 10. Note that an “ND” in a protein expression columnin Table 10 indicates that nothing was detected.

TABLE 10 Protein Expression of MSH1 And MSH2 in MSI-H Cancer SamplesTumor MSI Protein expression Sample Source Phenotype HMLH1 HMSH2 C172CHTN MSI-H − + C404 CHTN MSI-H − + C507 CHTN MSI-H − + C546 CHTN MSI-H− + C624 CHTN MSI-H ND ND C710 CHTN MSI-H − + C1166 CHTN MSI-H − + C5412CHTN MSI-H − + S15945 CHTN MSI-H + + A-1 Mayo Clinic MSI-H − + A-5 MayoClinic MSI-H − + A-7 Mayo Clinic MSI-H + − A-15 Mayo Clinic MSI-H − +A-19 Mayo Clinic MSI-H − + A-29 Mayo Clinic MSI-H − + A-49 Mayo ClinicMSI-H + − A-50 Mayo Clinic MSI-H − + A-73 Mayo Clinic MSI-H − + A-102Mayo Clinic MSI-H + − B-2 Mayo Clinic MSI-H − + B-52 Mayo Clinic MSI-H− + B-61 Mayo Clinic MSI-H − + B-75 Mayo Clinic MSI-H − + B-76 MayoClinic MSI-H − + B-93 Mayo Clinic MSI-H − + B-107 Mayo Clinic MSI-H − +B-155 Mayo Clinic MSI-H − + B-164 Mayo Clinic MSI-H − + B-166 MayoClinic MSI-H − + B-173 Mayo Clinic MSI-H − + B-199 Mayo Clinic MSI-H − +B-209 Mayo Clinic MSI-H − + B-210 Mayo Clinic MSI-H − + B-268 MayoClinic MSI-H − + B-299 Mayo Clinic MSI-H − + B-334 Mayo Clinic MSI-H − +B-379 Mayo Clinic MSI-H − + B-402 Mayo Clinic MSI-H − + B-564 MayoClinic MSI-H − +

EXAMPLE 6 MSI Multiplex Assay Development and Validation

Once the best loci were selected for use in designing multiplexes to beanalyzed according to the methods of the present invention, problemsassociated with multiplex PCR and incompatibility between loci needed tobe overcome. This required careful primer design and extensive trial anderror to find loci that were capable of simultaneous amplification usinga single set of PCR conditions. Problems encountered included: (1)primer-primer interactions that occurred when large number of oligoswere combined in a single PCR reaction, (2) primer design limitationsdue to sequence constraints at a particular locus (e.g., minimum size ofamplicon allowed by DNA sequence, sub-optimal % GC of primers,difficulty balancing Tm's for all primers under uniform PCR conditions,difficulty in finding primers with desirable thermal profiles tominimize non-specific amplification, hairpin formation and selfdimerization of primers, homology to other repeat sequences in humangenome), and (3) multiplex design allowing separation of all 9 lociwithin limited size range of 250 bp.

Based on extensive evaluation of close to 300 microsatellite markersdescribed in Examples 1 through 5, nine loci were selected for thepreferred MSI Multiplex Assay (Table 11). Three loci are monoplexrepeats (BAT-25, BAT-26 and MONO-15) and six were tetranucleotiderepeats (D1S518, D3S2432, D7S3046, D10S1426, D7S3070, D7S1808). Theseloci were found to be an excellent set of markers for identifying MSI intumor samples. Results of MSI analysis on 29 MSI-H and 30 MSI-L or MSScolon cancer samples using this nine-locus multiplex are summarized inFIG. 8.

A typical example of results produced from use of the nine locus MSIMultiplex described above is shown in FIG. 9. The image was generated bysimultaneous amplifying all nine selected microsatellite loci followedby separation of PCR products on an ABI 310 CE. Separation of all ninemicrosatellite loci in a single capillary (or gel lane) was accomplishedby designing the multiplex so that loci would not overlap in size orthrough use of different fluorescent dyes. The size ranges for thedifferent multiplex loci were determined by genotyping 93 samples fromAfrican-American individuals using MSI Multiplex described followingprotocol described below. In addition, a pooled Human Diversity DNAsample and control CEPH DNAs 1331-1 and 1331-2 (CORIELL Cell Repository)were included in the screening population. African-American samples wereused because they contain the greatest amount of genetic diversity foundin all racial groups.

TABLE 11 MSI Multiplex Assay Loci and Primers Re- Primer Primer GenBankpeat Size 1 (SEQ. 2 (SEQ. Locus ID No. Type Dye Range ID.) ID.) BAT-25U63834 Mono TMR 118-127 1 60 D10S1426 G08812 Tetra TMR 152-173 57 58D3S2432 G08240 Tetra TMR 198-234 17 59 BAT-26 U41210 Mono FL 103-116 6162 D7S3046 G10353 Tetra FL 122-163 55 56 D7S3070 G27340 Tetra FL 186-24953 54 MONO-15 AC007684 Mono JOE 115-117 7 8 D1S518 G07854 Tetra JOE136-178 49 50 D7S1808 G08643 Tetra JOE 190-218 51 52

Protocol for MSI Multiplex Assay. Template DNA from normal and tumortissues obtained from same individual were purified using QIAamp Bloodand Tissue Kit (QIAGEN, Santa Clarita, Calif.) following manufacturesprotocol. Two nanograms of template DNA in a 25 μl reaction volume wasPCR amplified using protocol detailed in Table 12, below, using thecycling profile described in Table 3, above.

TABLE 12 Amplification Mix for MSI Multiplex Assay Volume PCR Master MixComponent Per Sample Nuclease Free Water 17.00 μl GoldSTHR 10× Buffer(Promega) 2.50 μl Primer Pair Mix (10 μM) 2.50 μl AmpliTaq Gold DNAPolymerase (Perkin Elmer) 0.50 μl Template DNA (0.8 ng/μl) 2.50 μl TotalReaction Volume 25.00 μl

One microliter of PCR product was combined with 1 μl of Internal LaneStandard (Promega Corporation, Madison, Wis.) and 24 μl deionizedformamide. Samples were denatured by heating at 95° C. for 3 minutes andimmediately chilled on ice for 3 minutes. Separation and detection ofamplified fragments was performed on an ABI PRISM 310 Genetic Analyzerfollowing the standard protocol recommended in the User's Manual withthe following settings:

Run Module: GS STR POP4 (Filter set A) Injection Time:  4 secondsInjection Voltage: 15 kV Run Voltage: 15 kV Run Temperature: 60° C. RunTime: 24 minutes

Identification of normal and tumor allele amplicon sizes wasaccomplished by examining the appropriated electropherogram from the ABIPRISM 310 Genetic Analyzer and determining the predominant peaks foreach locus. One or two peaks or alleles were present for each locus innormal samples depending upon whether individual was homozygous orheterozygous for a particular marker. Allelic patterns or genotypes fornormal and tumor pairs were compared and scored as MSI positive if oneor more different alleles were present in the tumor DNA samples thatwere not found in normal sample from the same individual. Typicalexamples of results obtained using multiplex designed for MSI analysisare shown in FIGS. 10 and 11 for colon cancer and FIG. 12 for stomachcancer.

EXAMPLE 7 Amplification of DNA from PET Samples using MSI Multiplex

Microsatellite loci from the multiplex identified in Example 6, above,were evaluated for their ability to amplify DNA from formalin-fixedparaffin-embedded samples. DNA was extracted from three 10 micronsections cut from PET blocks using QIAamp Tissue Kit (Qiagen, SantaClarita, Calif.) according to the manufacture's instructions with thefollowing modifications. One hundred microliters of QIAGEN AE bufferpreheated to 70° C. was added to column, incubated for 5 minutes,centrifuged, then reapplied to column for second elution. Twomicroliters (out of 100 μl) of purified DNA solution was used astemplate for PCR reactions. The nine locus multiplexed primer setdescribed in Example 6 was used to amplify DNA from PET samples. Theresults indicate that the MSI Multiplex is capable of amplifying DNAfrom difficult and commonly used PET samples (FIG. 13).

EXAMPLE 8 Second MSI Multiplex System Configuration

A second set of nine loci was identified for analysis in a second MSImultiplex assay system, from eight of the best known set of lociidentified in Example 4, above, for determining MSI in colon tumorsamples, and from an additional mononucleotide repeat locus (BAT-40).Design of the multiplex loci was such that loci would not overlap insize within a single capillary or gel lane or could be labeled usingdifferent dyes when separated and overlap would occur. Using empiricallydetermined optimal experimental conditions, multiplex microsatelliteanalysis was performed on several hundred healthy individuals.

The set of nine loci selected for the MSI Multiplex Assay and relevantidentifying data for each locus is shown in Table 13, below.

TABLE 13 MSI Multiplex Assay Loci and Primers Re- Primer 1 Primer 2GenBank peat Size (SEQ. (SEQ. Locus ID No. Type Dye Range ID.) ID.)BAT-25 U63834 Mono TMR 118-127 1 60 D10S1426 G08812 Tetra TMR 152-173 5758 D3S2432 G08240 Tetra TMR 198-234 63 59 BAT-26 U41210 Mono FL 103-11661 62 D7S3046 G10353 Tetra FL 122-163 55 64 D7S3070 G27340 Tetra FL186-249 53 54 BAT-40 M38180 Mono JOE  81-105 65 66 MONO-15 AC007684 MonoJOE 145-155 67 68 D7S1808 G08643 Tetra JOE 190-218 51 52

Four of the loci in the second MSI multiplex are mononucleotide repeats(BAT-25, BAT-26, BAT-40 and MONO-15) and five are tetranucleotiderepeats (D3S2432, D7S1808, D7S3046, D7S3070, D10S1426). Table 13 showsprimer pairs used to amplify each locus in the second MSI multiplex, andapproximate size range of fragments produced upon amplification of eachlocus with each primer pair. The primer sequences identified in Table 13were selected as being particularly suitable for amplification of eachlocus in the second MSI multiplex.

Results of MSI analysis on 36 MSI-H and 30 MSS colon cancer samplesusing the nine-locus multiplex and primer pairs described in Table 13 isshown in Table 14, below, and illustrated in FIG. 14. FIG. 14 is a bargraph showing percent MSI measured at each locus of the multiplex fromeach of the two types of samples studied (i.e., MSI-H and MSS). Anadditional locus from the multiplex identified in Example 6, i.e.D1S518, was also included in this assay.

TABLE 14 MSI Multiplex Assay Results Locus % MSI-H % MSS D1S518 75 3BAT-25 100 0 D10S1426 86 7 D3S2432 86 3 BAT-40 100 0 MONO-15 100 0D7S1808 94 0 BAT-26 100 0 D7S3070 86 3 D7S3046 76 7

The results of the assay, summarized in Table 14 and illustrated in FIG.14, showed that amplification of BAT-40 resulted in clearerdiscrimination between MSI-H and MSS samples than D1S518. All nine lociamplified using the second MSI multiplex system were detected with veryhigh efficiency.

EXAMPLE 9 MSI Multiplex System Analysis

Ninety-three samples from African-American individuals, a pooled HumanDiversity DNA sample and control DNA from cell lines 1331-1 and 1331-2(Coriell Cell Repository, Camden N.J.) were examined using the ninemultiplex loci system described in Example 8. The simultaneousamplification of all nine selected microsatellite loci followed byseparation of products on an ABI 310 CE, as illustrated below, producedall expected products.

Protocol for MSI Multiplex Assay. Two nanograms of normal template DNAwere purified using QIAamp Blood and Tissue Kit (QIAGEN, Santa Clarita,Calif.) following the manufacturers protocol. DNA was amplified by PCRin a 25 μl reaction volume using protocol detailed in Table 15 and inTable 16, below.

TABLE 15 Amplification Mix for MSI Multiplex Assay Volume PCR Master MixComponent Per Sample Nuclease Free Water 17.00 μl GoldSTHR 10× Buffer(Promega) 2.50 μl Primer Pair Mix (10 μM) 2.50 μl AmpliTaq Gold DNAPolymerase (Perkin Elmer) 0.50 μl Template DNA (0.8 ng/μl) 2.50 μl TotalReaction Volume 25.00 μl

TABLE 16 Cycling profile for MSI Multiplex Assay  1 cycle 95° C. for 11minutes  1 cycle 96° C. for 1 minute 10 cycles 94° C. for 30 secondsramp 68 seconds to 60° C., hold for 30 seconds ramp 50 seconds to 70°C., hold for 90 seconds 20 cycles 94° C. for 30 seconds ramp 60 secondsto 60° C., hold for 30 seconds ramp 50 seconds to 70° C., hold for 90seconds  1 cycle 60° C. for 30 minutes  1 cycle Soak 4° C.

One microliter of PCR product was combined with 1 μl of Internal LaneStandard (DG2611, Promega Corporation, Madison, Wis.) and 24 μldeionized formamide. Samples were denatured by heating at 95° C. for 3minutes and immediately chilled on ice for 3 minutes. Separation anddetection of amplified fragments was performed on an ABI PRISM 310Genetic Analyzer following the standard protocol recommended in theUser's Manual with the following settings:

Run Module: GS STR POP4 (Filter set A) Injection Time:  4 secondsInjection Voltage: 15 kV Run Voltage: 15 kV Run Temperature: 60° C. RunTime: 24 minutes

Identification of normal allele amplicon sizes was accomplished byexamining the appropriate electropherogram from the ABI PRISM 310Genetic Analyzer and determining the predominant peaks for each locus.These results are shown in FIG. 15. One or two peaks or alleles werepresent for each locus in normal samples depending upon whetherindividual was homozygous or heterozygous for a particular marker.

EXAMPLE 10 MSI Multiplex System Analysis on Patient Samples

Allelic patterns or genotypes for paired normal and tumor tissues fromthe same individual were examined using the multiplex loci described inExample 8, above. The simultaneous amplification of all nine selectedmicrosatellite loci followed by separation of products on an ABI 310 CEshowed all expected products.

The protocol for the Multiplex MSI assay and the Cycling profile for thePatient samples were conducted exactly as in Example 9, above.Identification of normal and tumor allele amplicon sizes wasaccomplished by examining the electropherogram from the ABI PRISM 310Genetic Analyzer and determining the predominant peaks for each locus.

Typical results of the paired tissue examples are shown in FIG. 16. Oneor two alleles were present for each locus in normal samples dependingupon whether individual was homozygous or heterozygous for a particularmarker. Allelic patterns or genotypes for normal and tumor pairs werecompared and scored as MSI positive if one or more different alleleswere present in the tumor DNA samples that were not found in normalsample from the same individual.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

1. A method of analyzing microsatellite loci, comprising: a)co-amplifying at least three quasi-monomorphic mononucleotide repeatloci of human genomic DNA from at least one sample of genomic DNA in amultiplex amplification reaction comprising one or more primers, therebyproducing amplified DNA fragments; and b) determining the size of theamplified DNA fragments to detect the presence or absence ofmicrosatellite instability.
 2. The method of claim 1, wherein at leastone quasi-monomorphic repeat loci is selected from the group consistingof MONO-11, MONO-15, BAT-25, and BAT-26.
 3. The method of claim 1,wherein at least one quasi-monomorphic repeat loci comprises a perfectmononucleotide repeat comprising greater than 25 adenine residues. 4.The method of claim 1, wherein at least one quasi-monomorphicmononucleotide repeat loci comprises from 25 to 35 nucleotides.
 5. Themethod of claim 1, wherein the at least one sample of genomic DNAcomprises a first sample of genomic DNA originating from normalnon-cancerous biological material from an individual and a second sampleof genomic DNA originating from a tumor of the individual the methodfurther comprising: detecting microsatellite instability by comparingthe size of the amplified DNA fragments produced from co-amplifying thefirst sample of genomic DNA to the size of the amplified DNA fragmentsproduced from co-amplifying the second sample of genomic DNA.
 6. Themethod of claim 1, wherein the at least one sample of genomic DNA isfrom a tumor, further comprising evaluating microsatellite instabilityby comparing the fragment sizes of step (b) to the most commonlyobserved allele sizes for the at least three quasi-monomorphicmononucleotide repeat loci in a population.